Gelatin composition containing a halomethyl ketone of benzyloxycarbonyl phenylalanine

ABSTRACT

AQUEOUS SOLUTIONS OF GELATIN AND GELATIN-CONTAINING LAYERS SUCH AS PHOTOGRAPHIC SILVER HALIDE EMULSIONS CONTAINING BENZYLOXYCARBONYL PHENYLALANINE BROMOKETONE AND BENZYLOXYCARBONYL PHENYLALANINE BROMOMETHYL DETONE TO CONTROL ENZYME ACTIVITY AND DECELERATE BACTERIAL GROWTH AND REPRODUCTION. A PHENOLIC BIOSTAT MAY OPTIONALLY BE ADDED FOR IMPROVED RESULTS.

United States Patent O GELATIN COMPOSITION CONTAINING A HALO- METHYLKETONE F BENZYLOXYCARBONYL PHENYLALANINE Elliott N. Shaw, Shoreham,N.Y., assignor to Polaroid Corporation, Cambridge, Mass.

No Drawing. Original application May 22, 1972, Ser.

No. 255,709, now Patent No. 3,778,276. Divided and this application July9, 1973, Ser. No. 377,782

Int. Cl. C08h 7/00 US. Cl. 106-135 7 Claims ABSTRACT OF THE DISCLOSUREAqueous solutions of gelatin and gelatin-containing layers such asphotographic silver halide emulsions containing benzyloxycarbonylphenylalanine chloromethyl ketone and benzyloxycarbonyl phenylalaninebromomethyl ketone to control enzyme activity and decelerate bacterialgrowth and reproduction. A phenolic biostat may optionally be added forimproved results.

CROSS REFERENCE TO RELATED- APPLICATIONS This application is a divisionof US. Patent application Ser. No. 255,709 filed May 22, 1972 now US.Pat. 3,778,- 276.

BACKGROUND OF THE INVENTION activity and continued bacterial growth andreproduction is controlled.

DESCRIPTION OF THE PRIOR ART Photographic gelatin is a chemically stablesubstance when stored in a Warm aqueous emulsion under asepticconditions. However, an aqueous gelatin emulsion which is contaminatedwith pathogenic bacteria is rapidly degraded by what is known asbacterial action. In such bacterial action gelatin is not directlyconsumed by the bacteria because of the large size of gelatinspolypeptide molecules. Instead, the bacteria generate and inoculate thegelatin sol with a proteolytic enzyme which catalyzes the hydrolysis ofgelatin into smaller polypeptides, peptides,

and amino acids which are capable of passing through the bacterial cellwalls, thus supporting the intracellular metabolism necessary for theorganism growth and reproduction. Bacterial action causes seriousproblems in the pho tographic industry, particularly defects in thedeveloped image as it results in sensitized spots which becomespontaneously developable Without image exposure. In addition, bacterialaction can cause severe fog brought on by degradation of the gelatin.Degradation of gelatin also changes its viscosity and seriouslyinterferes with the anchoring characteristics of thin subbing layers.

It has been generally accepted that gelatin sols and photographicemulsions held for extended periods in cold storage before coatingrequire not only a depressant for slowing down the living processes oforganisms, but also a mechanism for destroying or inactivating theproteolytic enzymes already present or being produced by the stillviable organisms. If the enzymes are not destroyed or inactivated, theywill hydrolyze the gelatin sol and produce the other above-mentioneddeleterious results.

Unfortunately, most of the more frequently used emulsion biostats atpractical concentrations are apt to act 3,832,198 Patented Aug. 27, 1974Ice more as an enzyme preservative than a depressant. Thus, althoughbacterial activity is slowed, a minute quantity of enzyme already formedremains active. Pasteurization at 170180 F. destroys much of the enzymepotential but recontamination does occur and there are systems wherepasteurization is inconvenient or impractical.

Some of the compounds useful as gelatin preservatives in the past havebeen 1,3-diketones including highly enolized 1,3-diketones such asacetyl acetone; formaldehyde, phenol, thymol, esters of p-hydroxybenzoicacid, alcohols, and others as described in US. Pat. 2,226,183 of Staudet al., issued Dec. 24, 1940; British Pat. 987,010 of Du Pont issuedMar. 24, 1965; and British Pat. 968,883 of Gevaert issued Sept. 2, 1964.These compounds, however, are directed towards slowing down the livingprocesses of organisms and do very little in inactivating theproteolytic enzymes already present.

There are compounds known to be enzyme inhibitors such as iodoaceticacid and various fluorides such as sodium fluoride, potassium fluorideand potassium fluosilicate, but in permissible concentrations theiractivity was found to be low in certain gel sols and photographicemulsions and under some conditions they fail entirely, but they do notall function in an equivalent manner when incorporated into a gelatincomposition.

US. Pat. No. 3,503,746 discloses and claims the employment ofL-1-tosylamino-Z-phenethyl-chloromethyl-ketone (TPCK) in gelatinsolutions to control enzyme growth in gelatin solutions. However, theeffectiveness at given concentrations of TPCK in gelatin compositionsleaves something to be desired.

SUMMARY OF THE INVENTION It has now been found that benzyloxycarbonylphenylalanine chloromethyl ketone (ZPCK) and benzyloxycarbonylphenylalanine bromomethyl ketone (ZPBK) are highly effective ininhibiting the action of enzymes in an aqueous gelatin solution. ZPCKand ZPBK can be incorporated into gelatino-silver halide emulsions,gelatin interlayers and gelatin overcoats and will control enzymeactivity, thus inhibiting the degradation of gelatin and the attendantbacterial growth. The enzyme growth inhibition is accomplished withoutany adverse eflect on the photographic utility of the gelatincomposition. A phenolic biostat may also be employed with the enzymegrowth inhibitor.

DETAILED DESCRIPTION OF THE INVENTION ZPCK and ZPBK, halo methyl ketonesof benzyloxycarbonyl phenylalanine, have been found to be highlyeffective in inhibiting enzyme action in gelatin compositions whichresults in the degradation of the gelatin and obviates its fitness foremployment in, for example, photographic film units, particularlyphotographic silver halide emulsions. The employment of ZPCK and ZPBKhas been found to provide superior and unexpected results compared toconventional enzyme inhibitors for gelatin compositions. For example,compared to TPCK, referred to above, ZPCK and ZPBK show aneifectiveness, in terms of rate of enzyme inactivation, of as much astimes greater. In addition, ZPCK and ZPBK are efiective against enzymesresistant to the action of TPCK.

ZPCK and ZPBK may be employed satisfactorily over a relatively widerange. Preferably, the compounds are employed at a range of 25 to 300p.p.m. per gram of gelatin, more preferably 40 p.p.m. ZPCK and ZPBK areintroduced into the gelatin composition in suitable organic solvents, e.g., methanol or dimethyl sulfoxide.

ZPCK and ZPBK are known compounds and the methods of preparation may befound, for example, in Methods in Enzymology (C. H. W. Hirs, ed), p.684, Vol XI,

EXAMPLE I A cold solution of carbobenzoxy phenylalanine acid chloridefreshly prepared from 3.25 g. of carbobenzoxy- L-phenylalanine and 2.39g. of phosphorous pentachloride was added to the cold solution of diazomethane prepared according to the procedure set forth in page 250 ofOrg. Syn. Volume 4. After stirring the solution for 20 minutes, amixture of 16 milliliters of 32% hydrogen bromide in acetic acid, 22milliliters of 48% hydrogen bromide and 4.4 milliliters of water weradded and the mixture shaken vigorously. The ether layer was separated,washed with water, dried over anhydrous sodium sulphate and thenevaporated under a vacuum to a solid. The solid was recrystallized fromethanol to give 2 g. of White needles having a melting point of 97-99 C.of:

it Q-om-pn-c-om-m benzyloxy carbonyl phenylalanine bromomethyl ketone.

EXAMPLE II A cold diazo methane solution was added rapidly to a coldsolution of carbobenzoxy phenylalanine acid chloride freshly preparedfrom 6.8 g. of carbobenzoxy-L-phenylalanine and 5.64 moles ofphosphorous pentachloride in 150 milliliters of diethyl ether. Thereaction mixture was then stirred for 20 minutes at C. To the mixturewas then added 32 milliliters concentrated hydrochloric acid andmilliliters of water and stirring was continued for an additional 30minutes. The ether layer was then separated, washed with water anddilute sodium carbonate solution. The solution was then dried overanhydrous sodium sulphate, filtered and evaporated under a vacuum to anoil. The oil was recrystallized from 200 milliliters of ethanol andwater 1:1 ratio) to give 2 g. of white needles melting at 99-102 C. of:

benzoyloxycarbonyl phenylalanine chloromethyl ketone.

Conventional biostats, such as the phenolic biostats, e.g., phenol,thymol, p-chloro-meta-xylenol, and the like, may also be employed incombination with the enzyme inhibitors of the present invention in orderto obtain the combined effect of bacterial control to prevent enzymeproduction and the inhibition of enzyme attack on the gelatin.

The following non-limiting examples illustrate the employment of theenzyme inhibitors of the present invention.

Table 1 illustrates the effectiveness of the enzyme inhibitors of thepresent invention, as evaluated by the degree of viscosity change ofgelatin silver halide emulsions. Viscosity is measured in seconds at 40C. using an Ubbelohde tube.

TABLE 1 Days 0 7 14 21 28 35 42 Control 397 399 395 394 387 378 376 394397 396 395 390 375 377 300 p.p.m.lgm. gel ZPCK 389 339 389 393 392 392391 392 391 392 392 392 392 300 .m. 111. el ZPBK 391 391 392 392 392 392390 pp lg g 393 390 .392 392 392 392 390 The above table indicates thatsubstantially no viscosity change occurs employing the enzymeinhibitorsof the present invention.

The following experiment was designed to illustrate the inhibitingaction of ZPCK and ZPBK on an emulsion that has been inoculated with abad emulsion, i.e., one in which liquefaction and instability have beennoted visually. The ratio of bad to good was A; by weight.

TABLE 2 Beginning Signs of Liquefaction TPCK:

(500 p.p.m./g. gel, 5% gel emulsion) After 15 days. ZPBK:

(500 p.p.m./g. gel, 5% gel emulsion) After 35 days. (500 p.p.m./g. gel10% gel emulsion) After 38 days. ZPCK: k

(500 p.p.m./g. gel, 5% gel emulsion) Do. (500 p.p.m./g. gel 10% gelemulsion) After 42 days. Control: 1

(5% gel emulsion) After 3 days. (10% gel emulsion) After 5 days.

It will be noted that the enzyme inhibitors of the present invention arenot only highly effective, but are also significantly better than priorart inhibitors.

In some instances, the inhibitors of the present invention showedincreases in speed in emulsions, as compared with a control with noinhibitor. It should also be noted that both the dl and l isomers may beemployed equally satisfactorily.

As stated above, the enzyme inhibitors of the present invention may beemployed in any'aqueous gelatin composition, but are particularly usefulin gelatin-containing photographic elements, such as silver halideemulsions, dye image-forming material-containing layers, interlayers,and overcoats, including filter layers, spacer layers, and antihalationlayers.

Emulsions employing the enzyme inhibitors of the present invention maybe prepared by conventional methods and may include various adjuncts,for example, chemical sensitizing agents of U35. Pats. Nos. 1,574,944;1,623,499; 2,410,689; 2,597,856; 2,597,915; 2,487,850; 2,518,698;2,521,926; and the like; all according to the traditional procedures ofthe art, as described in Neblette, C. B., Photography Its Materials AndProcesses, 6th Ed., 1962.

Optical sensitization of the emulsions silverhalide crystals may beaccomplished by contact of the emulsion composition with an effectiveconcentration of the selected optical sensitizing dye dissolved in anappropriate dispersing solvent such as methanol, ethanol, acetone,water, and the like; all according to the traditional procedures of theart, as described in Hammer, F. M., The Cyanine Dyes And RelatedCompounds.

Additional optional additives, such as coating aids, hardeners,viscosity-increasing agents, stabilizers, preservatives, and the like,for example, those set forth hereinafter, also may be incorporated inthe emulsion formulation, according to the conventional procedures knownin the photographic emulsion manufacturing art.

The photoresponsive material of the photographic emulsion will, aspreviously described, preferably cornprise a crystal of ilver, forexample, one or more of the silver halides such as silver chloride,silver iodide, silver bromide, or mixed silver halides such as silverchlorobromide, silver chloroiodobromide or silver iodobromide, ofvarying halide ratios and varying silver concentrations.

As the binder for the respective emulsion strata, the aforementionedgelatin may be replaced in part with some other colloidal material suchas albumin; casein; or zein; or resins such as a cellulose derivative,as described in US. Pats. Nos. 2,322,085 and 2,327,808; polycarbonidesas described in US. Pat. No. 2,541,474; and vinyl polymers such as thosedescribed in an extensive multiplicity of readily available US. andforeign patents.

Photographic emulsions containing the enzyme inhibitor of the inventioncan also contain speed-increasing compounds such as quaternary ammoniumcompounds, polyethylene glycols or thioethers. Frequently, usefuleffects can be obtained by adding the aforementioned speedincreasingcompounds to the photographic developer solutions instead of, or inaddition to, the photographic emulsions;

Photographic elements containing the enzyme inhibitor of the instantinvention can be used in various kinds of photographic systems. Inaddition to being useful in X-ray and other non-optically sensitizedsystems, they can also be used in orthochromatic, panchromatic, andinfrared sensitive systems. The sensitizing addenda can be added tophotographic systems before or after any sensitizing dyes which areused.

Gelatin layers containing the inhibitor of the present invention mayalso be used in color photography, for example, in the color processedisclosed in US. Pat. No. 2,983,606, where the enzyme inhibitor, inaddition to being employed in the silver halide emulsion, is alsodisposed in the interlayers, including gelatin layers containing the dyedevelopers. The inhibitor is also useful in layers containing other dyeimage-forming materials such as those disclosed in US. Pats. Nos.2,647,049; 2,661,293; 2,698,244; 2,698,798; 2,802,735; 3,148,062;3,227,550; 3,227,551; 3,227,552; 3,227,554; 3,243,294; 3,330,655;3,347,671; 3,352,672; 3,364,022; 3,443,939; 3,443,940; 3,443,941;3,443,943; 2,774,668; and 3,087,817.

Silver halide emulsions containing the enzyme inhibitor of the inventioncan be used in diffusion transfer process which utilize the undevelopedsilver halide in non-image areas of the negative to form a positive bydissolving the undeveloped silver halide and precipitating it on asilver layer in close proximity to the original silver halide emulsionlayer. Such processes are described in US. Pats. Nos. 2,543,181 issuedFeb. 27, 1951 and 2,662,822 issued Dec. 15, 1953.

What is claimed is:

1. A composition comprising an aqueous solution of gelatin and, as anenzyme inhibitor, a halomethyl ketone of benzyloxycarbonylphenylalanine.

2. The composition as defined in claim 1 wherein said inhibitor isbenzyloxycarbonyl phenylalanine chloromethyl ketone.

3. The composition as defined in claim 1 wherein said inhibitor isbenzyloxycarbonyl phenylalanine bromomethyl ketone.

4. The composition as defined in claim 1 wherein said inhibitor ispresent at a level of ppm/gm. of gelatin.

5. The composition as defined in claim 1 wherein said inhibitor ispresent at a level of p.p.m./ gm. of gelatin.

6. The composition as defined in claim 1 wherein said aqueous solutioncontains a dye image-forming material disposed therein.

7. The composition of claim 1 which includes a phenolic biostat includedtherein.

References Cited UNITED STATES PATENTS 1,932,338 10/1933 Finkelstein96-109 1,946,057 2/ 1934 Britton 96-109 2,226,183 12/1940 1Staud 94l093,503,746 3/1970 McClintock 424-321 OTHER REFERENCES The Essentials ofHistidine in the Catalytic Action of Subtilism J. Biol. Chem. 243, 6312(1968), Elliott Shaw et al.

THEODORE MORRIS, Primary Examiner US. Cl. X.R.

